Facioscapulohumeral Muscular Dystrophy (FSHD) is a muscle wasting disease caused by genetic mutation in the subtelomeric region of chromosome 4. Healthy individuals have 11-100 repeated D4Z4 microsatellite units, whereas individuals with FSHD have 1-10 D4Z4 units. This genetic contraction makes the 4q35 chromosomal region more accessible to transcriptional machinery and permits transcription of nearby genes including DUX4, the proposed pathological agent in FSHD. DUX4 is a transcription factor that when aberrantly expressed induces transcription of many genes implicated in muscle development, the actin cytoskeleton, regulation, apoptotic signaling pathways, and germline development. While many potential biomarkers of FSHD, including TRIM43, MBD3L2, ZSCAN4, and PITX1, all of which are downstream of DUX4, have been identified from studies of muscle biopsy and of muscle cells in vitro, the pathophysiological role of DUX4 and these target genes has yet to be studied in a humanized in vivo model. The D4Z4 contraction is not replicable in animal models, nor do the effects of Dux4 expression in murine muscle cells replicate those seen in human cells. To overcome these problems, the laboratory has developed a method of xenografting human-derived muscle precursor cells, isolated from patients with FSHD and healthy controls, into the tibialis anterior compartment of immune-deficient mice. These cells grow, fuse, and mature into force-producing human muscle fibers within the mouse hind limb. These human muscle fibers can be studied in intact grafts or following dissociation and isolation in culture. This novel in vivo FSHD model will be used to study DUX4 and its potential downstream gene targets, TRIM43, MBD3L2, ZSCN4, and PITX1. The ultimate goal is to determine how their patterns of expression and localization in relation to DUX4 within individual fibers lead to dystrophy ? experiments that have never been performed in an FSHD model system before. Further analyses will attempt to reveal the primary defect in FSHD muscle, leading to the creation of targeted FSHD therapies. This study hypothesizes that DUX4 expression in a small percentage of the nuclei of mature human muscle fibers is responsible for activating DUX4 gene targets in the same or nearby myonuclei and that together these induce local muscle wasting, leading to the phenotype seen in FSHD. The hypothesis will be tested by i) comparing the molecular phenotype of intact muscle as well as fibers isolated and cultured from FSHD-derived xenografts to controls and by ii) examining the functional phenotype of FSHD-derived xenografts compared to controls. Molecular methods such as qRT-PCR, immunofluorescent labeling, and confocal microscopy combined with electrophysiology-based functional measures will be employed to explore the phenotype in living muscle from FSHD xenografts. This study overcomes the limitations of current models of FSHD associated with viral vectors or incomplete development in culture, as it examines the pathologic consequences of endogenous DUX4 expression and its downstream targets in mature human muscle fibers grown within the mouse hind limb.